The Role And Mechanism Of PI3K Signaling Pathway On Treg Homeostasis In Sarcoidosis And Type 1 Diabetes

The first part: Selective inhibition of PI3 K rebalancesTh1/Th17/Treg in mouse model of pulmonary sarcoidosis.Beckgroud: Sarcoidosis is a multi-system immune disease of unknown etiology and the pathological manifestations were non-caseating granulomas in different organs,mainly involving the lungs.Studies have demonstrated that the pathogenesis of sarcoidosis is related to Th1/Th17/Treg disorders.PI3K/Akt pathway is critical for maintaining Treg function and homeostasis,which is abnormally activated in patients with sarcoidosis.In our previous study,we found that inhibition of all the four type Ⅰ PI3 K subunits with BKM120 can more effectively induce Treg homeostasis than selective inhibition of PI3 K α,β and δ subunits with LY294002,suggesting that the level and function of Treg in sarcoidosis may be more dependent on the p110γ subunit.Aim: In this study,we selected two specific inhibitors of PI3 K subunit,AS-605240(specific inhibition of PI3Kp110γ subunit)and CAL-101(specific inhibition of PI3Kp110δ subunit),to explore whether specific intervention of PI3Kp110γ subunit and PI3Kp110δ subunit can slow down the development of sarcoidosis by restoring the immunoregulatory capacity of Treg.Method: We established a sarcoidosis mouse model by Sod A immunostimulation.The second immune stimulus was accompanied by the inhibitor’s treatment.Activation of PI3 K signaling pathway was detected by immunofluorescence and Western blot.CD4+CD25+ T cells were separated by MACS immunomagnetic beads and the proportion of Th1,Th2,Th17,Treg and the immunosuppressive function of Treg were detected by FACS.The expression of related m RNAs was detected by real-time quantitative PCR to evaluate the regulatory effects of CAL-101 and AS-605240 on Th1/Th2,Th17/Treg cells in sarcoidosis mice and their effects on the proliferation,differentiation and function of Treg.Results: In the sarcoidosis mouse model,the decrease of Treg level and the loss of Treg’s function are accompanied by the activation of PI3K/Akt signaling pathway.CAL-101 was more effective in alleviating sarcoidosis granuloma development than AS-605240.Specifically,CAL-101 can inhibit the abnormal activation of PI3K/Akt in Treg,induce Th1/Th17/Treg rebalancing,and enhance the function of Treg.AS-605240 inhibit the abnormal activation of PI3K/Akt signaling in Treg,but the effect on inducing Th1/Th17/Treg rebalancing and enhancing Treg function is not as obvious as CAL-101.Conclusion: Combined with the results of previous studies,our results suggest a synergistic effect of the p110δ and γ subunits in restoring the immunoregulatory capacity of Treg,thereby slowing the development of sarcoidosis.This may provide a theoretical basis for clinical search for targeted drugs and immunomodulatory therapy to control sarcoidosis activities.The second part: Inhibition of PI3 K signaling pathway combined with syngeneic bone marrow transplantation in the treatment of type 1 diabetes in mouse models.Beckgroud: T1 D is an autoimmune disease mediated by lymphocytes and characterized by immune islet inflammation and selective β cell damage.Our previous study found that syngeneic bone marrow transplantation(syn-BMT)in new-onset(10 days after onset of diabetes)T1D mice could effectively reverse the status of diabetes,accompanied by increasing CD4+CD25+Foxp3+ Treg cells in the spleen,while syn-BMT had poor effects onlong-onset(20 or 40 days after onset)T1DAim: We performed syn-BMT on streptozotocin(STZ)-induced long-onset T1 D mice(20 days after onset)and intervened with PI3 K inhibitors to explore whether inhibition of the PI3 K signaling pathway could restore the immunoregulatory capacity of Treg,so as to improving the effect of syn-BMT in the treatment of T1 D with a longer onset time.Method: We established a T1 D mouse model by multiple low-dose STZ injections.Twenty days after the onset of diabetes,syn-BMT was performed and BKM120 was used to intervene in mice at the same time,or BKM120 was used to intervene stem cells in vitro and then reinfused.Observation was performed for 126 days after transplantation,and fasting blood glucose changes and glucose tolerance were recorded to observe the therapeutic effects.Samples were collected 126 days after transplantation,and FACS,q-PCR,and MACS immunomagnetic bead sorting CD4+CD25+ T cells were used to detect PI3 K inhibitors on the Th1/Th2 and Treg/Th17 cell subsets in the T1 D mouse model after syn-BMT.The effects on the proliferation,differentiation and function of Treg cells were also tested.At the same time,H&E,Masson and PAS staining were used to detect the effects of PI3 K inhibitor combined with hematopoietic stem cell transplantation on the morphology of pancreas and kidney in T1 D mice.Results: We found that inhibiting PI3K/Akt signaling pathway by BKM120,combined with syn-BMT,enhanced Treg’s immunosuppressive function and balanced Th17/Treg,and regulated blood glucose in T1 D mice.PI3 K inhibitors improve the therapeutic effects of syn-BMT on T1 D mice with a longer onset time,and have a certain therapeutic effect on diabetic nephropathy.Conclusion: The results suggest that targeting PI3 K signaling pathway can improve the therapeutic effects of syn-BMT on T1 D mice with a longer onset time.This study may provide a theoretical basis for the clinical application of HSCT in T1 D.